Compositions and methods for delivery of genetic material

ABSTRACT

A soluble ionic complex is formed by an aqueous mixture of a benzylammonium group-containing surfactant and a polynucleic acid sequence. When the mixture forms a vesicular complex, the sequence is packaged therein. This composition is useful in pharmaceutical compositions and in methods of delivering the polynucleic acid sequence (which preferably encodes a protein or peptide) to a cell for expression. Such methods are useful in therapy, as vaccines and as gene therapy agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a 371 of PCT/US98/22841, which claims the benefit of thepriority of U.S. patent application Ser. No. 60/063,360, filed Oct. 28,1997.

FIELD OF THE INVENTION

The present invention relates to compositions and methods forintroducing genetic material into cells. More particularly, the presentinvention relates to compositions and methods for in vitro and in vivogene transfer, which can be used to deliver protective and/ortherapeutic agents including genetic material that encodes proteintargets for immunization and therapeutic proteins.

BACKGROUND OF THE INVENTION

The direct introduction of a normal, functional gene into a livinganimal has been studied as a means for replacing defective geneticinformation. DNA can be introduced directly into cells of a livinganimal using viral vectors, liposomes, lipid complexes, ligand/DNAconjugates, and microprojectile bombardment, among other methods.Various methods and compositions for mediating transfer of DNA to cellsin vivo and/or in vitro are referred to in U.S. Pat. No. 5,593,972,issued Jan. 14, 1997; U.S. Pat. No. 5,580,859, issued Dec. 3, 1996; U.S.Pat. No. 5,589,466 issued Dec. 31, 1996; U.S. Pat. No. 5,676,954, issuedNov. 19, 1996; International Patent Publications Nos. WO90/11092,published Mar. 21, 1990; WO93/17706, published Mar. 10, 1993;WO93/23552, published May 21, 1993; and WO94/16737, published Jan. 26,1994, and the priority applications cited therein.

Despite the knowledge extant in the art, there remains a need forimproved methods of DNA transfer, as well as for improved methods andcompositions for in vivo and in vitro nucleic acid transfer. Thereremains a need for improved methods of drug delivery.

SUMMARY OF THE INVENTION

In one aspect, the invention provides soluble, ionic complex comprisingan aqueous mixture of a benzylammonium group-containing surfactant and apolynucleic acid sequence. In one embodiment, the complex is avesicular-like or liposomal-like complex comprising an aqueous mixtureof a benzylammonium group-containing surfactant of the formula describedherein and a polynucleic acid sequence, with the sequence substantiallypackaged in the vesicular complex.

In another aspect, the invention provides a mixture of multiple ionicand/or vesicular complexes of uniform size, as above described. In oneembodiment, the composition is formed by mixing an aqueous solution of abenzylammonium-containing surfactant, preferably benzalkonium chloride,with a polynucleic acid sequence.

In still another aspect, the invention provides a pharmaceuticalcomposition comprising at least one, and preferably multiple ioniccomplexes or vesicular complexes described above and a suitablepharmaceutical carrier.

In yet another aspect, the invention provides a method of introducing apolynucleic acid sequence into a cell comprising the step of contactingsaid cell with the above described complexes or compositions containingthem.

In another aspect, the invention provides a method of facilitating theuptake of a polynucleic acid sequence into a cell comprising contactingthe cell with a soluble ionic complex described above, or with apolynucleic acid substantially packaged in a vesicular complex formed byan aqueous mixture of a benzylammonium-containing surfactant with thepolynucleic acid sequence.

In a further aspect, the invention provides methods of inducing animmune response in a mammalian or vertebrate subject to a pathogenicantigen or disease, which methods include the step of administering tocells of said subject, an effective amount of a complex as describedherein, wherein the polynucleic acid sequence encodes at least oneepitope that is identical or substantially similar to an epitope of aantigen of said pathogen, or a sequence encoding a target protein, saidprotein comprising an epitope identical or substantially similar to anepitope of a protein associated with cells that characterize saiddisease. The epitope or protein-encoding sequence is under the controlof regulatory sequences that direct expression of said protein in thecells of said subject.

In still another aspect, the invention provides a method of treating amammalian or vertebrate subject for a disease comprising the step ofadministering to cells of said subject, an effective amount of acomposition comprising a complex of this invention formed by an aqueousmixture of a benzylammonium-containing surfactant and a polynucleic acidsequence, wherein said polynucleic acid sequence comprises a sequencewhich encodes a protein that produces a therapeutic effect on thesubject or a protein that compensates for a missing, non-functional orpartially functioning native mammalian protein, the protein-encodingsequence under the control of regulatory sequences that directexpression of said protein in the cells of said subject.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the humoral (antibody) response measuredin optical density (OD) at 450 nm in serum of individual Balb/C mice toan aqueous composition of the invention containing the indicatedconcentrations of benzalkonium chloride and indicated amounts of a DNAplasmid encoding the gD₂ protein of Herpes Simplex Virus as measured bystandard ELISA. The positive and negative controls are the same plasmidwith no transfection facilitating agent (DNA only) and the plasmid withno gD₂ encoding sequence with no transfection facilitating agent(023ctrl). Each bar represents a single animal. See Example 4 below.

FIG. 2 is a bar graph showing group average humoral (antibody) responsesin the animals of FIG. 1. The responses are measured according toAntibody Response Calculations in ng/ml, as defined in Example 4 below.

FIG. 3 is a scatter plot graph showing the individual (animalsrepresented by ⋄, □, Δ, X, and *) and group average (represented by )cellular responses of the animals of FIG. 1. Systemic cellular response(SI) was measured using a splenic cell proliferation assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel soluble ionic complex comprisingan aqueous mixture of a benzylammonium group-containing surfactant; anda polynucleic acid sequence. These soluble complexes may be in the formof vesicular complexes containing polynucleic acid sequencessubstantially packaged therein. Compositions containing the complexesare useful as pharmaceutical (therapeutic and vaccinal) agents and asgene therapy reagents in methods for introducing the polynucleic acidsequences into a cell for a variety of therapeutic and prophylacticpurposes, as well as for research purposes. The compositions and methodsof the present invention provide for a high level of uptake and functionof the polynucleic acid sequences and molecules.

I. The Soluble Ionic Complexes

A soluble ionic complex of this invention is formed by an aqueousmixture of a benzylammonium group-containing surfactant and apolynucleic acid sequence. The combination of the surfactant and thepolynucleic acid sequence forms a vesicular-like or liposomal-likestructure, in which substantially all of the polynucleic acid sequencebecomes packaged. Minor amounts of the polynucleic acid sequence areassociated with the exteriors of the vesicular complex. Without wishingto be bound by theory, these complexes appear to provide in vivostability to the polynucleic acid sequences associated therewith, andthus facilitate transfection of such sequences into host cells.

A. The Surfactant

The benzylammonium group-containing surfactant is preferably asurfactant of the formula:

wherein X is an anion;

each R¹ is independently a hydrogen or a lower alkyl group comprisingfrom 1 to 6 carbon atoms;

R² is CH₂ or —O—;

R³ is H, CH₃, C₂H₅, phenyl, mono-substituted phenyl, or di-substitutedphenyl, wherein said substitutions are independently selected from amongC₁-C₁₀ branched or straight chain alkyls groups; and

n is an integer of 2 through 7, provided that when n is 1, R³ is methyl,ethyl, phenyl or substituted phenyl; when n is 4 through 6, R³ is H,methyl, ethyl or phenyl; when n is 6, R³ is H, methyl or ethyl; and whenn is 7, R³ is H or methyl.

Examples of preferred benzylammonium-group containing surfactantsinclude those which comprise a dimethyl benzyl ammonium group linked toan alkyl group or an alkyl group linked to an aromatic group. The anionis selected from among anions that results in soluble complexes with thepolynucleic acid sequence in water. In some embodiments, the anion is ahalide, a sulfate, or a carbonate. In one preferred embodiment, theanion in the surfactant is a halide, such as chloride. One of skill inthe art may select from among a number of suitable anions for thepreparation of a surfactant suitable for the present invention.

One presently preferred example of a benzylammonium group-containingsurfactant is a benzalkonium halide, such as benzalkonium chloride.Benzalkonium chloride is a cationic surfactant known to condense DNA [V.Jelen et al, Journal of Electroanalytical Chemistry, 377:197-203(1994)]. It has also been used as an antimicrobial agent for parenteralpreparations [Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field; and “Pharmaceutical Dosage Forms:Parenteral Medication”, Vol.I, K. Avis et al (eds), Marcel Dekker, Inc.,New York (1992)]. Benzalkonium chloride refers to commercially availablesurfactants which are a mixture of alkyldimethylbenzylammonium chloridesof the formula above where [CH₂CH₂R²]_(n)—R³ is a mixture of alkylsC₈H₁₇ to C₁₈H₃₇, i.e., n is 2-6, CH₂ and R³ is hydrogen, methyl orethyl. Benzalkonium chloride which may be used as the surfactant in thecomplexes, compositions, kits and methods of the invention may behomogenous or may contain a mixture of compounds characterized by havingtwo or more different R groups according to the formula above.Benzalkonium chloride, U.S.P. grade can be purchased from SpectrumChemical Mfg. Corp., Gardena, Calif.

Another exemplary surfactant useful in this invention is a benzethoniumhalide, such as benzethonium chloride. Benzethonium chloride refers to acommercially available surfactant N,N-dimethyl-N-[2- [2-[4(1,1,3,3-tetramethylbutylphenoxy)ethoxy]ethyl]ammonium chloride, and isdescribed in U.S. Pat. Nos. 2,115,250, 2,170,111 and 2,229,024. It hasalso been used as an antimicrobial agent for parenteral preparations[Pharmaceutical Dosage Forms, cited above]. Benzethonium chloride hasthe formula set out above where n is 2 and R³ is 4-[1,1,3,3-tetramethylbutyl]phenyl. Benzethonium chloride, U.S.P. grade can be purchasedfrom Spectrum Chemical Mfg. Corp., Gardena, Calif.

The complexes of the present invention comprise a final concentration ofbenzylammonium-group containing surfactant between about 0.001 to about2.4% by volume (w/v). Desirably, the complexes have a finalconcentration of the surfactant of between about 0.001-0.10% w/v. Morepreferably, the concentration of surfactant in the complexes is betweenabout 0.005-0.06% w/v. A particularly desirable vesicular complex ofthis invention contains a benzylammonium-group containing surfactant ina concentration of between about 0.005-0.03%w/v. Manipulation of theother components of the complex, e.g., the polynucleic acid, and thebuffering agents and optional isotonicity and pH adjusting agents, canreduce the toxicity of the surfactant, and permit a soluble complex tobe formed at a variety of concentrations of surfactant withoutencountering precipitation. One of skill in the art given theinstructions provided herein is expected to be able to readilymanipulate the components of this invention to provide such solublecomplexes at a variety of surfactant concentrations.

B. The Polynucleic Acidc Sequence

Another component of the complexes of the present invention is apolynucleic acid sequence, which when admixed with the above-describedaqueous surfactant, forms a soluble ionic complex therewith. Inpreferred embodiments, the polynucleic acid sequence becomessubstantially packaged in the vesicular-like or liposomal-like complex,and only a minor amount of such polynucleic acid sequence is associatedwith the exterior of the complex.

The polynucleic acid sequences which form part of the complexes of thisinvention are preferably “dissociated from an infectious agent”, i.e.,are not part of a viral, bacterial or eukaryotic vector, either active,inactivated, living or dead, that is capable of infecting a cell. Insome embodiments, the polynucleic acid sequence present in compositionsof the present invention are preferably free from infectious agents suchas viral particles, particularly retroviral particles, and arepreferably non-infectious plasmid DNA molecules. In some preferredembodiments, the compositions are free of lipids, such as cationiclipids, and/or other surfactants, and/or local anaesthetics. In someembodiments, the polynucleic acid sequences are free from theprecipitating agent CaPO₄.

The complexes and compositions of the present invention preferablycomprise between about 10 μg/ml to about 20 mg/ml of polynucleic acidsequences or molecules. Preferably, the aqueous compositions ofsurfactant and polynucleic acid sequences which form the complexes ofthe invention comprises a concentration of polynucleic acid sequences ofbetween about 50 μg/ml to about 10 mg/ml of polynucleic acid sequencesor molecules. In other preferred embodiments, the aqueous compositionsof surfactant and polynucleic acid sequences which form the complexes ofthe invention comprises a concentration of polynucleic acid sequences ofbetween about 100 μg/ml to about 1 mg/ml of polynucleic acid sequencesor molecules.

For example, one embodiment of the complexes of the inventions containsabout 0.1-5.0 mg/ml polynucleic acid in a final concentration of0.010-0.030% w/v benzylammonium-group containing surfactant. Somepreferred embodiments comprise 0.010% w/v benzylammonium-containingsurfactant and 0.1 mg/ml polynucleic acid molecules. Other preferredembodiments comprise 0.010% w/v benzylammonium-group containingsurfactant with about 0.5 mg/ml nucleic acid molecules. Still otherpreferred embodiments comprise 0.020% w/v benzylammonium-groupcontaining surfactant with about 0.5 mg/ml nucleic acid molecules.

A particularly desirable embodiment of the complexes of the presentinvention is formed by between about 100-500 μg DNA molecules at aconcentration of 0.1-0.5 mg/ml in a final concentration of 0.010-0.030%w/v benzalkonium chloride or benzethonium chloride. Another preferredembodiment comprises 0.010% w/v benzalkonium chloride and 0.1 mg/mlnucleic acid molecules. Still other preferred embodiments comprise0.010% w/v benzalkonium chloride and about 0.5 mg/ml nucleic acidmolecules. Some preferred embodiments comprise 0.020% w/v benzalkoniumchloride and 0.5 mg/ml nucleic acid molecules. Some preferredembodiments comprise 0.010% w/v benzethonium chloride and 0.1 mg/mlnucleic acid molecules. Some preferred embodiments comprise 0.010% w/vbenzethonium chloride and 0.5 mg/ml nucleic acid molecules. Somepreferred embodiments comprise 0.020% w/v benzethonium chloride and 0.5mg/ml nucleic acid molecules.

The polynucleic acid sequence of this invention may be any nucleic acidsequence and may take a variety of known forms, as taught elsewhere inthe art. Thus, as used herein, the terms “polynucleic acid sequence”,“nucleic acid molecule”, “polynucleotide”, “DNA construct”, “geneticconstruct” and “nucleotide sequence” are interchangeable. Polynucleicacid sequences of this invention can be deoxyribonucleic acid sequences(DNA) and/or ribonucleic acid sequences (RNA). These nucleic acidsequences or molecules may be cDNA, genomic DNA, synthesized DNA, DNAmolecules or plasmids or a hybrid thereof, or an RNA molecule such asmRNA. The polynucleic acid sequence may also encode antisense sequenceswhich inhibit gene expression of genes whose expression is undesirable.A polynucleic acid molecule may serve as a template for antisensemolecules and ribozymes and such sequences may be preferably linked toregulatory elements necessary for production of sufficient copies of theantisense and ribozyme molecules encoded thereby respectively or aribozyme.

Polynucleic acid sequences or molecules useful in the present inventionmay serve a variety of functions, but are essentially provided to aselected host cell for a multitude of known therapeutic, prophylactic,and research uses. For example, the sequences are useful in thecomplexes of the invention as: 1) sequences encoding for proteins thatfunction as prophylactic and/or therapeutic immunizing agents; 2)replacement copies of defective, missing or non-functioning genes; 3)sequences encoding therapeutic proteins; 4) antisense sequences orsequences encoding for antisense molecules; or 5) sequences encodingfor, or genetic templates for, ribozymes.

Thus, in desired embodiments, the polynucleic acid sequence or moleculemay comprise a sequence that encodes a peptides or protein. The sequencemay be a plasmid which comprises a nucleotide sequence that encodes aprotein or peptide, the encoding sequence operably linked to regulatorysequences directing expression of the protein or peptide in a host cell.Such regulatory sequences direct replication, transcription, translationand/or expression of the encoded protein or peptide in selected hostcells, e.g., mammalian or vertebrate cells. As used herein, the term“expressible form” refers to polynucleic acid sequences or geneconstructs which contain the necessary regulatory elements operablylinked to a coding sequence that encodes a target protein, such thatwhen present in the host cell, the coding sequence will be expressed.

The regulatory elements necessary for expression of a sequence encodinga protein or peptide include a promoter (constitutive or inducible), aninitiation signal or codon, a termination signal or stop codon, and apolyadenylation signal. In addition, enhancers are often required, aswell as other sequences, e.g., a Kozak region, etc. Such regulatoryelements may be selected from among those known to be preferred in aselected host cell, and the polynucleic acid sequence may likewisecontain codons which are known to be preferentially expressed in certainhost cells. Such regulatory elements are operable in the cell of amammalian or vertebrate subject or tissue to whom they are administered.Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thehost cells. Examples of promoters useful to practice the presentinvention, especially in the production of a genetic vaccine or genetherapy vector, include but are not limited to, promoters from SimianVirus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, HumanImmunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR)promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMVimmediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus(RSV) as well as promoters from human genes such as human actin, humanmyosin, human hemoglobin, human muscle creatine and humanmetallothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego, Calif.), referred toas the SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the polynucleic acid sequence ofthe complex. Such additional elements include enhancers, such as thoseselected from the group including but not limited to: human actin, humanmyosin, human hemoglobin, human muscle creatine and viral enhancers suchas those from CMV, RSV and EBV. The polynucleic acid sequences of thisinvention may also include a mammalian origin of replication in order tomaintain the construct extrachromosomally and produce multiple copies ofthe construct in a mammalian or vertebrate cell. Plasmids pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

Additionally, polynucleic acid sequence (e.g., DNA) which is useful topromote integration of the polynucleic acid sequence into the chromosomeof the cell may also be included in the polynucleic acid sequence or DNAmolecule useful in this invention. One embodiment of a polynucleic acidsequence is as a linear minichromosome including a centromere, telomeresand an origin of replication.

The polynucleic acid sequence may also contain an additional elementwhich serves as a target for cell destruction if it is desirable toeliminate cells receiving the sequence for any reason. A herpesthymidine kinase (tk) gene in an expressible form can be included in thepolynucleic acid molecule of the complex. Upon administration of thedrug gangcyclovir, any cell transfected with the complex and thusproducing tk, will be selectively killed. Thus, the polynucleic acidsequence can provide the means for the selective destruction of cellstransfected therewith.

Depending on the use to which the complex is applied, the polynucleicacid sequence may encode a wide variety of peptides or proteins usefulin pharmaceutical reagents and in research. As one example, the proteinsand/or peptides encoded by the polynucleic acid sequence of thevesicular complex can include a target protein useful to induce orelicit a therapeutic or prophylactic immune response. The target proteinis an immunogenic protein which shares at least an epitope with aprotein from the pathogen (e.g., a virus, a bacterium, yeast, parasite,etc) or from an undesirable cell-type such as a cancer cell or a cellinvolved in autoimmune disease against which immunization is required.The protein can be an epitope identical or substantially similar to anepitope of a antigen of the pathogenic microorganism or undesirable celltype. As used herein, the term “substantially similar epitope” is meantto refer to an epitope that has a structure which is not identical to anepitope of a protein but nonetheless invokes a cellular or humoralimmune response which cross reacts to that protein. The protein can bean epitope identical or substantially similar to an epitope of a proteinassociated with, e.g., hyperproliferating cells; or an epitope identicalor substantially similar to an epitope of a protein associated with,e.g, cells that characterize an autoimmune disease.

The polynucleic acid sequence may also encode a therapeutic orcompensating protein, i.e., it can encode a protein or peptide which cancompensate for a protein product that is deficient, missing,nonfunctional or partially functioning, endogenously produced, in a cellor mammalian or vertebrate subject due to an absent, defective,non-functioning or partially functioning endogenous gene. Thepolynucleic acid sequence can also encode a protein or peptide thatproduces a therapeutic effect in a mammalian or vertebrate subject.

Exemplary protein products can readily be selected by one of skill inthe art for insertion into a host cell. Among the non-exclusive lists ofprotein-encoding polynucleic acid sequences are sequences from anoncogene selected from the group consisting of myb, myc,fyn, ras, sarc,neu and trk. The sequences can also encode a protein product of thetranslocation gene bcl/abl; a protein product of P53; or for example,the protein EGRF. Still other exemplary polynucleic acid sequencesuseful in various aspects of this invention can encode a variable regionof an antibody made by a B cell lymphoma; a variable region of a T cellsurface receptor of a T cell lymphoma; a variable region of an antibodyinvolved in B cell mediated autoimmune disease; and a variable region ofa T cell surface receptor involved in T cell mediated autoimmunedisease.

Thus, any polynucleic acid sequence which is desired to be inserted in aselected host cell can form part of the vesicular-like or liposomal-likecomplex of the present invention. One of skill in the art oftherapeutics, vaccines and gene therapy may readily select andincorporate a desired polynucleic acid sequence using the teachings ofthe present invention.

C. The Buffer and Other Reagents in the Complex

The aqueous mixture of the benzylammonium-containing surfactant and thepolynucleic acid sequence which form the soluble ionic complexes orvesicular complexes of the invention may also contain other optionalagents, such as aqueous buffering agents, isotonicity adjusting agents,and pH adjusting agents. Suitable buffers for use in forming thecomplexes may be conventionally selected from among many known buffersused in the formation of pharmaceutical products. Among a non-exclusivelist of buffers are phosphate buffers, such as phosphate buffered salineand citrate buffers. Selection of such buffers is clearly within theskill of the art. Preferably, the aqueous mixture which forms thecomplexes contains a buffer in a concentration of about 2 to about 50mM, preferably about 5 to 30 mM. In one embodiment, the compositioncontains about 5 mM phosphate buffered saline.

The compositions of the present invention may also preferably containisotonicity adjusting agents. For example, for pharmaceuticalcompositions for parenteral administration, especially intramuscularly,subcutaneously and intradermally, the aqueous mixture forming thecomplexes is desirably isotonic. However, as desired, one of skill inthe art may readily make the compositions hypotonic or hypertonic. Someexamples of-typical tonicity adjusting agents include, withoutlimitation, sodium chloride, sucrose, mannitol, sorbitol, and trehalose.For example, where the complex is desirably hypotonic to isotonic, antonicity adjusting agent, e.g., sucrose, is present in the aqueousadmixture forming the complex in a concentration of 0 to about 9.25%w/v. For example, where the complex is desirably hypertonic, an tonicityadjusting agent, e.g., sucrose, is present in the aqueous admixtureforming the complex in a concentration of greater than 9% w/v. One ofskill in the art of pharmaceutical preparation can readily adjust thischaracteristic of the complex.

Similarly, ionic strength of the complex may be adjusted by one of skillin the art by the addition of charged molecules, such as sodiumchloride. In the examples below, sodium chloride is present at aconcentration of between about 0-0.9% w/v.

The aqueous mixture forming the complexes of this invention arepreferably characterized by a pH between about 6.0 to about 8.0. Morepreferably, a desirably pH is about 6.7±0.5. Suitably pH adjustments maybe readily made by a selection of agents and is well within theknowledge of one skilled in the pharmaceutical arts.

D. Examples of Complexes of the Invention

According to one embodiment of the invention, a soluble ionic complex isformed by an isotonic, aqueous admixture benzalkonium chloride andplasmid DNA, such as illustrated in Examples 1 and 4 below. In anotherexample of the invention illustrated below, the complex of thisinvention is formed from an aqueous, isotonic mixture of benzethoniumchloride and plasmid DNA, as illustrated in Example 5. Such compositionscomprise the polynucleic acid sequence substantially packaged in thevesicular complex formed by an aqueous mixture of abenzylammonium-containing surfactant and the sequence, as demonstratedby Examples 2 and 3. Similarly, other compositions of this inventioncontain multiple vesicular complexes of uniform size, each vesicularcomplex containing polynucleic acid sequence substantially packaged inthe complexes formed by admixing an aqueous mixture of thebenzylammonium-containing surfactant and a solution containing thepolynucleic acid sequence. The method of preparing such complexes of theinvention is described in detail in Example 1 below.

The compositions of this invention, i.e., the complexes formed by thebenzylammonium-group containing surfactants and polynucleic acidsequences, increase and/or facilitate uptake and/or expression of thepolynucleic acid sequences by host cells, compared to the uptake orexpression which occurs when the identical polynucleic acid sequence ormolecule is administered to a host cell in the absence of thebenzylammonium-group containing surfactants. See the results of Example3 below.

II. Pharmaceutical Compositions of this Invention

The complexes and compositions of this invention may be employed inpharmaceutical compositions and in methods to introduce polynucleic acidsequences, e.g., genetic material, into cells in vitro or in vivo. Apharmaceutical composition of this invention comprises the soluble ioniccomplexes as described above in a suitable pharmaceutical carrier. Insome instances, the aqueous buffer may itself be suitable. The vaccinesand therapeutics according to the present invention are formulatedaccording to the mode of administration to be used. One having ordinaryskill in the art can readily formulate a pharmaceutical composition thatcomprises a complex as described above. In cases where intramuscularinjection is the chosen mode of administration, an isotonic formulationis preferably used. Generally, additives for tonicity can include sodiumchloride, dextrose, mannitol, sorbitol and lactose. In some cases,isotonic solutions such as phosphate buffered saline are preferred.Stabilizers include gelatin and albumin. In some embodiments, avasoconstriction agent is added to the formulation. The pharmaceuticalpreparations according to the present invention are provided sterile andpyrogen free. Although for pharmaceutical use, any route ofadministration may be employed, it is preferred that the composition ofthe invention be an injectable formulation. In one embodiment of theinvention a pharmaceutical composition comprises a vesicular complexwhich contains the polynucleic acid molecule substantially packaged inthe vesicle, with some minor amount of the sequence associated with theexterior of the vesicular complex, with the complex being in a suitablepharmaceutical carrier.

The compositions of the present invention, when used as pharmaceuticalcompositions, can comprise about 1 ng to about 1000 μg of DNA. In somepreferred embodiments, the vaccines and therapeutics contain about 10 ngto about 800 μg DNA. In some preferred embodiments, the vaccines andtherapeutics contain about 0.1 to about 500 μg DNA. In some preferredembodiments, the vaccines and therapeutics contain about 1 to about 350μg DNA. In some preferred embodiments, the vaccines and therapeuticscontain about 25 to about 250 μg DNA. In some preferred embodiments, thevaccines and therapeutics contain about 100 μg DNA.

In addition, other agents which may function as transfecting agentsand/or replicating agents and/or inflammatory agents and which may beco-administered with the benzylammonium-group containingsurfactants-nucleic acid molecule complexes include growth factors,cytokines and lymphokines such as alpha-interferon, gamma-interferon,platelet derived growth factor (PDGF), colony stimulating factors, suchas G-CSF, GM-CSF, tumor necrosis factor (TNF), epidermal growth factor(EGF), and the interleukins, such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10and IL-12. Further, fibroblast growth factor, surface active agents suchas immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant,LPS analog including monophosphoryl Lipid A (MPL), muramyl peptides,quinone analogs and vesicular complexes such as squalene and squalene,and hyaluronic acid may also be used administered in conjunction withthe compositions of the invention.

The compositions of the present invention may be combined with collagenas an emulsion and delivered parenterally. The collagen emulsionprovides a means for sustained release of DNA. Preferably 50 μl to 2 mlof collagen are used. About 100 μg DNA are combined with 1 ml ofcollagen in a preferred embodiment using this formulation. Othersustained release formulations such as those described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield. Such formulations include aqueous suspensions, oil solutions andsuspensions, emulsions and implants as well as reservoirs andtransdermal devices. In some embodiments, time release formulations forcompositions of the present invention are provided. In some embodiments,it is preferred that the compositions of the present invention are timereleased between 6-144 hours, preferably 12-96 hours, more preferably18-72 hours.

III. Methods of Use of the Complexes

A. In Vitro Transfection Methods

The invention provides in vitro transfection methods using the complexesand compositions comprising the aqueous mixtures of polynucleic acidsequences and benzylammonium-group containing surfactants as abovedescribed. A method of the invention facilitates the uptake of apolynucleic acid sequence into a cell comprising the step of contactingthe cell with a soluble ionic complex comprising an aqueous mixture of abenzylammonium group-containing surfactant and a polynucleic acidsequence, as described above. According to this in vitro method, thecomplex may be introduced into tissue cultures or cells in solution inthe form of a vesicular complex in which the polynucleic acid sequenceis substantially packaged in the complex, with a minor amount ofsequence associated with the exterior of the complex.

B. Methods of In Vivo Administration

The invention also provides in vivo therapeutic, prophylactic and genetherapy methods of transferring polynucleic acid sequences (e.g.,genetic material) into cells of a mammal or a vertebrate using thepharmaceutical compositions and complexes of this invention. Thebenzylammonium-group containing surfactants are administered as amixture with the nucleic acid molecule. In preferred embodiments, thebenzylammonium-group containing surfactants are mixed with nucleic acidmolecules to form vesicular complexes. The methods of this inventioninvolve the step of administering to a cell or tissue of the mammal orvertebrate, a composition comprising a benzylammonium-group containingsurfactant and a polynucleic acid sequence. Transfection of thepolynucleic acid sequence, e.g., the DNA or RNA molecule in thesurfactant:polynucleic acid sequence complex, into a living cell resultsin the expression of the DNA or RNA. Where the DNA or RNA encode adesired protein, the desired protein is produced. When taken up by acell, the nucleotide sequence encoding the desired protein operablylinked to the regulatory elements may remain present in the cell as afunctioning extrachromosomal molecule or it may integrate into thecell's chromosomal DNA. The complex may be introduced into cells and thepolynucleotide may remains as separate genetic material in the form of aplasmid. Alternatively, the complex can be employed to introduce alinear polynucleic acid sequence (DNA) into the cell, which DNA canintegrate into the chromosome. When introducing the complex into thecell, reagents which promote DNA integration into chromosomes may beadded.

i. Inducing Immune Responses and Therapeutic Treatment

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual against a pathogen, allergen or abnormal,disease-related cell. In one embodiment, the invention provides a methodof inducing an immune response in a mammalian or vertebrate subject,preferably a human, to a pathogenic antigen comprising the step ofadministering to cells of said subject, an effective amount of acomposition or soluble complex as described above. In this complex, thepolynucleic acid sequence comprises a sequence which encodes at leastone epitope that is identical or substantially similar to an epitope ofan antigen of the pathogen against which an immune response can begenerated which will be directed against the target pathogen antigen,allergen or antigen of an abnormal and/or disease-related cell. Thepolynucleic acid sequence can also encode a peptide or protein which isimmunologically cross reactive to the target pathogen antigen, allergenor antigen of an abnormal and/or disease-related cell. Theepitope-encoding sequence, which is part of the polynucleotide sequence,is under the control of regulatory sequences that direct expression ofsaid protein in the cells or tissue of the mammalian or vertebratesubject.

In a similar embodiment, the invention provides a method of immunizing amammalian or vertebrate subject against a disease comprising the step ofadministering to said subject a composition comprising an effectiveamount of a composition comprising a complex of this invention, wherethe polynucleic acid sequence comprises a nucleotide sequence encoding atarget protein, operatively linked to regulatory sequences directing theexpression of said protein in the cells of said subject. The targetprotein can be an epitope identical or substantially similar to anepitope of a protein associated with cells that characterize thedisease.

In other aspect, the method of the invention permits therapeutictreatment of a mammalian or vertebrate subject for a disease comprisingthe step of administering to cells of said subject, an effective amountof a composition comprising a complex described above. In the complexesuseful in this aspect of the invention, the polynucleic acid sequencecomprises a sequence which encodes a protein or peptide that produces atherapeutic effect on the subject, said protein-encoding sequence underthe control of regulatory sequences that direct expression of saidprotein in the cells of said subject.

According to these aspects of the present invention, the DNA or RNA thatencodes a desired protein is introduced into the cells of an individualwhere it is expressed, thus producing the desired protein. In suchembodiments, an immune response is generated that is immunologicallycross reactive with a pathogen antigen, allergen or antigen of theabnormal and/or disease-related cell. The resulting immune response isbroad based: in addition to a humoral immune response, immune responsesfrom both arms of the cellular immune response are elicited. The methodsof the present invention are useful for conferring prophylactic andtherapeutic immunity. Thus, a method of immunizing includes both methodsof protecting an individual from pathogen challenge, or occurrence orproliferation of specific cells as well as methods of treating anindividual suffering from pathogen infection, hyperproliferative diseaseor autoimmune disease with which the target protein is associated.

This aspect of the method of the present invention is useful to immunizeindividuals against pathogenic agents and organisms such that an immuneresponse against a pathogen protein provides protective immunity againstthe pathogen. The present invention is useful to combathyperproliferative diseases and disorders such as cancer by eliciting animmune response against a target protein that is specifically associatedwith the hyperproliferative cells. The present invention is useful tocombat autoimmune diseases and disorders by eliciting an immune responseagainst a target protein that is specifically associated with cellsinvolved in the autoimmune condition.

In some preferred embodiments related to immunization applications, thegenetic construct contains nucleotide sequences that encode a targetprotein and further include genes for proteins which enhance the immuneresponse against such target proteins. Examples of such genes are thosewhich encode cytokines and lymphokines such as those listed above inPart II. In some embodiments, it is preferred that the gene for B7.2and/or GM-CSF is included in genetic constructs used in immunizingcompositions.

The present invention may be used to immunize an individual against allpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated, such as viruses, and prokaryote such as gonorrhoeae,listeria and shigella. In addition, the present invention is also usefulto immunize an individual against protozoan pathogens which include astage in the life cycle where they are intracellular pathogens. As usedherein, the term “intracellular pathogen” is meant to refer to a virusor pathogenic organism that, for at least part of its reproductive orlife cycle, exists within a host cell and therein produces or causes tobe produced, pathogen proteins. One of skill in the art, given thisdisclosure can readily select viral families and genera, or pathogensincluding prokaryotic and eukaryotic protozoan pathogens as well asmulticellular parasites, for which vaccines according to the presentinvention can be made. See, e.g., the tables of such pathogens ingeneral immunology texts and in U.S. Pat. No. 5,593,972. In somepreferred embodiments, the methods of immunizing an individual against apathogen are directed against HIV, HTLV or HBV.

Because DNA and RNA are both relatively small and can be producedrelatively easily, the present invention provides the additionaladvantage of allowing for vaccination with multiple pathogen antigens.The polynucleic acid sequence used in a composition such as a geneticvaccine employing a complex of this invention can include geneticmaterial which encodes many pathogen antigens. For example, severalviral genes may be included in a single construct thereby providingmultiple targets. In addition, multiple inoculants which can bedelivered to different cells in an individual can be prepared tocollectively include, in some cases, a complete or, more preferably, anincomplete such as a near complete set of genes in the vaccine. Forexample, a complete set of viral genes may be administered using twoconstructs which each contain a different half of the genome which areadministered at different sites. Thus, an immune response may be invokedagainst each antigen without the risk of an infectious virus beingassembled. This allows for the introduction of more than a singleantigen target and can eliminate the requirement that protectiveantigens be identified.

Another aspect of the present invention provides a method of conferringa broad based protective immune response against hyperproliferatingcells that are characteristic in hyperproliferative diseases and to amethod of treating individuals suffering from hyperproliferativediseases. In such methods, the introduction of complexes of thisinvention serves as an immunotherapeutic, directing and promoting theimmune system of the individual to combat hyperproliferative cells thatproduce the target protein. As used herein, the term “hyperproliferativediseases” is meant to refer to those diseases and disorderscharacterized by hyperproliferation of cells. Examples ofhyperproliferative diseases include all forms of cancer and psoriasis.It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell-associated protein” into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. As used herein, the term “hyperproliferativeassociated protein” is meant to refer to proteins that are associatedwith a hyperproliferative disease. To immunize againsthyperproliferative diseases, a complex of the invention that includes apolynucleic acid sequence which encodes a protein that is associatedwith a hyperproliferative disease is administered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF.

In addition to oncogene products as target antigens, target proteins foranti-cancer treatments and protective regimens include variable regionsof antibodies made by B cell lymphomas and variable regions of T cellreceptors of T cell lymphomas which, in some embodiments, are also usedtarget antigens for autoimmune disease. Other tumor-associated proteinscan be used as target proteins such as proteins which are found athigher levels in tumor cells including the protein recognized bymonoclonal antibody 17-1A and folate binding proteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer inindividual. Using genetic screening and/or family health histories, itis possible to predict the probability a particular individual has fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies. T cell mediated autoimmune diseases includeRheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmunethyroiditis, reactive arthritis, ankylosing spondylitis, scieroderma,polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener'sgranulomatosis, Crohn's disease and ulcerative colitis. Each of thesediseases is characterized by T cell receptors that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases. Vaccination against the variable region of the Tcells would elicit an immune response including CTLs to eliminate thoseT cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vα-17 and Vα-17. Thus, vaccination with a DNAconstruct that encodes at least one of these proteins will elicit animmune response that will target T cells involved in RA [Howell, M. D etal, Proc. Natl. Acad. Sci. USA, 88:10921-10925 (1991); Paliard, X. etal., Science, 253:325-329 (1991); Williams, W. V. et al., J. Clin.Invest., 90:326-333 (1992)]. In MS, several specific variable regions ofTCRs which are involved in the disease have been characterized. TheseTCRs include Vβ-7 and Vα-10. Thus, vaccination with a DNA construct thatencodes at least one of these proteins will elicit an immune responsethat will target T cells involved in MS [Wucherpfennicf, K. W., et al.,Science, 248:1016-1019 (1990); Oksenberg, J. R., et al., Nature,345:344-346 (1990)]. In scieroderma, several specific variable regionsof TCRs which are involved in the disease have been characterized. TheseTCRs include Vβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15,Vα-16, Vα-28 and Vα-12. Thus, vaccination with a DNA construct thatencodes at least one of these proteins will elicit an immune responsethat will target T cells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines using thecomplexes of this invention can be prepared using this information.

B cell mediated autoimmune diseases include Lupus (SLE), Graves disease,myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody. In order to treat patients suffering from a B cell mediatedautoimmune disease, the variable region of the antibodies involved inthe autoimmune activity must be identified. A biopsy can be performedand samples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation. For example, in the case of SLE, one antigen is believed tobe DNA. Thus, in patients to be immunized against SLE, their sera can bescreened for anti-DNA antibodies and a vaccine can be prepared whichincludes DNA constructs that encode the variable region of such anti-DNAantibodies found in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. (1987) Sequence of Proteins ofimmunological Interest U.S. Department of Health and Human Services,Bethesda Md. In addition, a general method for cloning functionalvariable regions from antibodies can be found in Chaudhary, V. K., etal., Proc. Natl. Acad. Sci. USA, 87:1066 (1990).

In some embodiments of the invention, the individual is subject to asingle vaccination to produce a full, broad immune response. In someembodiments of the invention, the individual is subject to a series ofvaccinations to produce a full, broad immune response. According to someembodiments of the invention, at least two and preferably four to fiveinjections are given over a period of time. The period of time betweeninjections may include from 24 hours apart to two weeks or longerbetween injections, preferably one week apart. Alternatively, at leasttwo and up to four separate injections are given simultaneously atdifferent sites.

In some embodiments of the invention, a complete vaccination includesinjection of a single inoculant which contains a compositions of thisinvention which includes a polynucleic acid sequence including sequencesencoding one or more targeted epitopes.

In some embodiments of the invention, a complete vaccination includesinjection of two or more different inoculants into different sites. Forexample, in an HIV vaccine according to the invention, the vaccinecomprises two inoculants in which each one comprises compositions ofthis invention encoding different viral proteins. This method ofvaccination allows the introduction of as much as a complete set ofviral genes into the individual without the risk of assembling aninfectious viral particle. Thus, an immune response against most or allof the virus can be invoked in the vaccinated individual. Injection ofeach inoculant is performed at different sites, preferably at a distanceto ensure no cells receive both genetic constructs. As a further safetyprecaution, some genes may be deleted or altered to further prevent thecapability of infectious viral assembly.

ii. Gene Therapy

Other aspects of the present invention relate to gene therapy; that is,to compositions for and methods of introducing nucleic acid moleculesinto the cells of an individual exogenous copies of genes which eithercorrespond to defective, missing, non-functioning or partiallyfunctioning genes in the individual or which encode therapeuticproteins, i.e., proteins whose presence in the individual will eliminatea deficiency in the individual and/or whose presence will provide atherapeutic effect on the individual thereby providing a means ofdelivering the protein by an alternative means from proteinadministration. In aspects of the invention relating to gene therapy,constructs with origins of replication including the necessary antigenfor activation are preferred. Thus, a method of treating a mammalian orvertebrate subject for a disease comprises administering to cells ofsaid subject, an effective amount of a composition comprising a complexas above-described. The polynucleic acid sequence of the complex whichis useful in this method comprises a sequence which encodes a proteinthat compensates for a missing, non-functional or partially functioningnative mammalian protein, said protein-encoding sequence under thecontrol of regulatory sequences that direct expression of said proteinin the cells of said subject.

In some of the embodiments of the invention that relate to gene therapy,the gene constructs contain either compensating genes or genes thatencode therapeutic proteins. Examples of compensating genes include agene which encodes dystrophin or a functional fragment, a gene tocompensate for the defective gene in patients suffering from cysticfibrosis, an insulin, a gene to compensate for the defective gene inpatients suffering from ADA, and a gene encoding Factor VIII. Examplesof genes encoding therapeutic proteins include genes which encodeserythropoietin, interferon, LDL receptor, GMCSF, IL-2, IL-4 and TNF.Additionally, genetic constructs which encode single chain antibodycomponents which specifically. bind to toxic substances can beadministered.

In some preferred embodiments, the dystrophin gene is provided as partof a mini-gene and used to treat individuals suffering from musculardystrophy. In some preferred embodiments, a mini-gene which containscoding sequence for a partial dystrophin protein is provided. Dystrophinabnormalities are responsible for both the milder Becker's MuscularDystrophy (BMD) and the severe Duchenne's Muscular Dystrophy (DMD). InBMD dystrophin is made, but it is abnormal in either size and/or amount.The patient is mild to moderately weak. In DMD no protein is made andthe patient is wheelchair-bound by age 13 and usually dies by age 20. Insome patients, particularly those suffering from BMD, partial dystrophinprotein produced by expression of a mini-gene delivered according to thepresent invention can provide improved muscle function.

In some preferred embodiments, genes encoding IL-2, IL-4, interferon orTNF are delivered to tumor cells which are either present or removed andthen reintroduced into an individual. In some embodiments, a geneencoding gamma interferon is administered to an individual sufferingfrom multiple sclerosis.

Antisense molecules and ribozymes may also be delivered to the cells ofan individual by introducing genetic material which acts as a templatefor copies of such active agents. These agents inactivate or otherwiseinterfere with the expression of genes that encode proteins whosepresence is undesirable. Constructs which contain sequences that encodeantisense molecules can be used to inhibit or prevent production ofproteins within cells. Thus, production proteins such as oncogeneproducts can be eliminated or reduced. Similarly, ribozymes can disruptgene expression by selectively destroying messenger RNA before it istranslated into protein. In some embodiments, cells are treatedaccording to the invention using constructs that encode antisense orribozymes as part of a therapeutic regimen which involves administrationof other therapeutics and procedures. Gene constructs encoding antisensemolecules and ribozymes use similar vectors as those which are used whenprotein production is desired except that the coding sequence does notcontain a start codon to initiate translation of RNA into protein. Insome embodiments, it is preferred that the vectors contain an origin ofreplication and an expressible form of the appropriate nuclear antigen.

Ribozymes are catalytic RNAs which are capable of self-cleavage orcleavage of another RNA molecule. Several different types of ribozymes,such as hammerhead, hairpin, Tetrahymena group I intron, ahead, andRNase P are known in the art [S. Edgington, Biotechnology, 10:256-262(1992)]. Hammerhead ribozymes have a catalytic site which has beenmapped to a core of less than 40 nucleotides. Several ribozymes in plantviroids and satellite RNAs share a common secondary structure andcertain conserved nucleotides. Although these ribozymes naturally serveas their own substrate, the enzyme domain can be targeted to another RNAsubstrate through base-pairing with sequences flanking the conservedcleavage site. This ability to custom design ribozymes has allowed themto be used for sequence-specific RNA cleavage [G. Paolella et al., EMBOJ., 1913-1919 (1992)]. It will therefore be within the scope of oneskilled in the art to use different catalytic sequences from varioustypes of ribozymes, such as the hammerhead catalytic sequence and designthem in the manner disclosed herein. Ribozymes can be designed against avariety of targets including pathogen nucleotide sequences and oncogenicsequences. Certain preferred embodiments of the invention includesufficient complementarity to specifically target the abl-bcr fusiontranscript while maintaining efficiency of the cleavage reaction.

iii. Routes of Administration

In any of the above described pharmaceutical methods, the complex may beadministered by any suitable route for such therapy. Among such routesare included parenteral routes, such as intramuscular, intraperitoneal,intradermal, subcutaneous, intravenous, intraarterial, intraoccular, andintrathecal routes of administration. Mucosal routes of administrationare also useful, including rectal, vaginal, urethral and intranasalroutes. Topical and transdermal administration is also useful forcompositions and methods of this invention. Administration by inhalationis also useful. Suppository preparations or other appropriate dosageforms are also useful. Oral administration may also be employed in themethods of this invention. Preferred routes of administration includeintramuscular, intraperitoneal, intradermal and subcutaneous injection.

Compositions of this invention may be administered by means including,but not limited to, traditional syringes, needleless injection devices,or “microprojectile bombardment gene guns”. According to someembodiments of the present invention, the complex of this invention issimultaneously administered to an individual intradermally,subcutaneously and intramuscularly using a needleless injection device.Needleless injection devices are well known and widely available. Onehaving ordinary skill in the art can, following the teachings herein,use needleless injection devices to deliver compositions of thisinvention to cells of an individual. Needleless injection devices arewell suited to deliver compositions of this invention to all tissue.They are particularly useful to deliver compositions of this inventionto skin and muscle cells, intradermally, subcutaneously andintramuscularly. In some embodiments, a needleless injection device maybe used to propel a liquid that contains the surfactant:DNA complexestoward the surface of the individual's skin. The liquid is propelled ata sufficient velocity such that upon impact with the skin the liquidpenetrates the surface of the skin, permeates the skin and muscle tissuetherebeneath. In some embodiments, a needleless injection device may beused to deliver compositions of this invention to tissue of other organsin order to introduce a nucleic acid molecule to cells of that organ.

According to methods of this invention, complexes or compositions ofthis invention may be administered directly into the individual to beimmunized. By any route, the compositions of this invention areintroduced into cells which are present in the body of the individual.Delivery of the polynucleic acid sequences which encode target proteinscan confer mucosal immunity in individuals immunized by a mode ofadministration in which the material is presented in tissues associatedwith mucosal immunity. Thus, in some examples, the complex of thisinvention is delivered by administration in the buccal cavity within themouth of an individual, or administered rectally, vaginally, or to theurethra.

Alternatively, the compositions may be introduced by various means exvivo into removed cells of the individual which are reimplanted afteradministration. Such means include, for example, ex vivo transfection,electroporation, microinjection and microprojectile bombardment. Afterthe complex of the invention is taken up by the cells, the cells arereimplanted into the individual. It is contemplated that otherwisenon-immunogenic cells that have the polynucleic acid sequencesincorporated therein can be implanted into the individual even if thevaccinated cells were originally taken from another individual.

In some embodiments, the compositions of the present invention compriseas the polynucleic acid sequence an attenuated viral vaccine that may bedelivered as a genetic construct. Such constructs may allow forproduction of viral particles. Delivery of the attenuated vaccine as apolynucleic acid sequence in a complex of this invention allows for aneasier way to produce large quantities of safe, pure active immunizingproduct.

In some embodiments, the compositions of the present invention may beadministered with or without the use microprojectiles. In someembodiments, the compositions of the present invention may be deliveredto the cells of an individual free of solid particles. As used herein,the phrase “free of solid particles” is meant to refer to a liquid thatdoes not contain any solid microprojectile used as a means to perforate,puncture or otherwise pierce the cell membrane of a cell in order tocreate a port of entry for compositions of this invention into the cell.For example, the compositions of the present invention are administeredby means of a microprojectile particle bombardment procedure as taughtby Sanford et al. in U.S. Pat. No. 4,945,050 issued Jul. 31, 1990. Insome embodiments of the invention, the compositions of the presentinvention are administered as part of a liposome complex.

The methods of the present invention are useful in the fields of bothhuman and veterinary medicine. Accordingly, the present inventionrelates to genetic immunization of mammals, and vertebrates, such asbirds and fish. The methods of the present invention can be particularlyuseful for mammalian species including human, bovine, ovine, porcine,equine, canine and feline species.

The following examples illustrate the preferred methods for preparingthe benzylammonium-containing surfactant/polynucleic acid complexes ofthe present invention and further illustrate that such compositionsfacilitate the uptake of the polynucleic acid. These examples whichemploy as the surfactant, benzalkonium chloride or benzethoniumchloride, and as the polynucleic acid sequences, plasmid sequencescontaining a herpes simplex virus gD gene merely illustrate selectionsof the surfactant, the polynucleic acid sequence, the type of sequenceand source of the sequence. It is understood by one of skill in the art,that other selections for these components of the invention may bereadily selected as taught by this specification. These examples areillustrative only and do not limit the scope of the invention.

EXAMPLE 1 A Benzylammonium-Containing Surfactant/Polynucleic AcidFormulation of The Invention

A composition containing uniform ionic, vesicular complexes withpolynucleic acid packaged in an aqueous benzylammonium-containingsurfactant is formulated according to this invention as follows. Forthis example, the polynucleic acid sequence component is plasmid DNA,and the benzylammonium-containing surfactant, benzylammonium chloride.The buffer used is phosphate buffer. Other conventional buffers, such ascitrate buffer, also can be used instead of phosphate buffer. Tonicityof the resulting solution may be adjusted with sodium chloride, sucrose,other conventionally known isotonic agents, such as mannitol, sorbitol,trehalose, or any non-ionic agents from the list in Remington'sPharmaceutical Sciences, supra.

An illustrative formulation of this invention is prepared by admixingunder conditions of ambient temperature the components listed in TableI. Generally, a stock solution of the benzylammonium-containing compoundis prepared in the selected buffer buffer. A polynucleic acid solution,e.g., a DNA or RNA solution is prepared containing the selectedconcentration of polynucleic acid sequence, e.g, plasmid DNA, in theselected buffer with the tonicity adjusting agent. Before admixture,both solutions are preferably filtered conventionally, for example,using a 0.22 μm Millex GV syringe filter. Suitable amount of thesurfactant solution is added to suitable volumes of the polynucleic acidby slow mixing. The desired concentration is made by selecting theconcentration of surfactant solution and a desired concentration of thepolynucleic acid solution.

A composition according to this invention must be soluble. The endpointof concentrations of the components of the complex is generallyprecipitation. It is preferred that the charge ratio of thebenzylammonium-containing surfactant and the polynucleic acid be lessthan 1, and that excess positive charge be avoided. The conditions ofthe solution, and the amounts of polynucleic acid sequence andsurfactant can be manipulated to increase solubility and reduce thetoxicity of the surfactant concentrations. Desirably, the pH of theadmixture is between 6 and 8, and more preferably between 6.2 and 7.2.The desired isotonicity, hypotonicity (<0.9% w/v NaCl or equivalent) orhypertonicity (>0.9% w/v NaCl or equivalent), can be adjusted bytonicity adjusting agents [see, e.g., Remington's, cited above].

Table I provides an illustrative composition of the invention. Otherranges of the components of the soluble ionic complex of the inventionare disclosed above.

TABLE I Component Range Plasmid DNA   10 μg/ml-20.0 mg/ml Benzylammoniumcontaining surfactant    0.001-2.4% w/v Buffer (e.g. phosphate)     2-30mM Sodium chloride     0-0.9% w/v Tonicity adjusting agent (e.g.,sucrose)     0-13% w/v

EXAMPLE 2 Scanning and Transmission Electron Microscopic Studies withBenzalkonium Chloride:DNA Complexes

Scanning electron microscopy (SEM) and transmission electron microscopy(TEM) were employed to visualize the resulting complexes formed by thebenzylammonium-containing surfactant and polynucleic acid compositionsof this invention.

A. Preliminary Studies

Electron microscope studies were made of the following benzalkoniumchloride/DNA complex formulations of the invention:

a) 0.025% benzalkonium chloride/0.5 mg/mL of DNA,

b) 0.0125% benzalkonium chloride/0.5 mg/mL DNA,

c) 0.025% benzalkonium chloride/0.1 mg/mL of DNA,

d) 0.012% benzalkonium chloride/0.05 mg/mL of DNA. These formulationswere made in the same manner as described for Example 1, except that thebenzalkonium chloride concentrations are different as stated above. Eachformulation containing 8.71% sucrose w/v and 5 mM phosphate buffer atpH6.7±0.5.

Electron microscopic photographs (not pictured) illustrated that eachformulation formed uniform vesicular complexes indistinguishable fromthose derived from classical liposomes and cationic liposomes.

B. Additional SEM and TEM Studies

i. Formulations of this invention were prepared by mixing DNA at 0.5mg/mL, with benzalkonium chloride (0.02% w/v) in 10 mM citrate orphosphate buffers, pH 6.7 (±0.5), and 50 mM NaCl, substantially asdescribed above.

The structures visible by both SEM and TEM are indistinguishable fromthose derived from classical liposomes and cationic liposomes. The sizeof these particles varied from 50 nm to 230 nm. However, most structureshad an uniform size distribution ranging from 70 to 100 nm.

ii. Additional studies were performed by mixing DNA at 5 mg/ml in higherconcentrations of benzalkonium chloride, up to 0.04% w/v, in 10 mMcitrate or phosphate buffers, pH 6.7 (±0.5), and 50 mM NaCl,substantially as described above. Benzalkonium chloride alone does notform vesicular complexes in water. At higher than ≧0.1% w/v,benzalkonium chloride alone is soluble and forms micelles. However, uponaddition of DNA, vesicular complexes are formed, when observed by SEM.The vesicular complexes range in size from 50 (at 0.01% benzalkoniumchloride) to 400 nm particles (0.04% w/v benzalkonium chloride).Benzalkonium chloride at concentrations higher than ≧0.04% w/v underthese conditions, in the presence of DNA formed a fine precipitate thatcontained DNA (determined by agarose gel electrophoresis of theprecipitate). At concentrations above 0.04% benzalkonium chloride underthese conditions, the precipitates were snowy and flocculent.

These observations and the SEM and TEM pictures described herein (notpictured) show that benzalkonium chloride:DNA compositions according tothis invention form a vesicular-like or liposomal-like structure. Thequaternary ammonium cationic head group of the benzalkonium chloride ispositively charged independent of protonation, and forms complexes withDNA, that decrease its solubility. At benzalkonium chlorideconcentrations lower than 0.04% w/v in the compositions above, thecomplexes remain soluble, while at benzalkonium chloride concentrations≧0.04% w/v in the compositions above, the hydrophilicity of thesecomplexes is reduced to near neutrality. The association of DNA with theprecipitates further indicates complexation.

iii. SEM was carried out on formulations that contained two differentconcentrations of DNA (100 μg/ml and 0.5 mg/ml), and fixedconcentrations of benzalkonium chloride (0.02% w/v). Similar particledistribution was found at both concentrations of DNA, but in largernumbers with higher DNA concentrations. Larger particles in higher DNAconcentrations appear to have derived from the fusion of several 50 nmparticles. Formation of larger particles was dependent on theconcentration of DNA in the formulation. These particles were visualizedby shadow casting in SEM analysis using carbon, and were visible byshort (10 second) exposure to the contrasting agent uranyl acetate inTEM analysis. The TEMs show membranous structures typical of liposomesdescribed in literature.

As controls, SEM of DNA alone and benzalkonium chloride alone in aqueoussolutions were performed. Vesicular structures are found only whenaqueous solutions of DNA and benzalkonium chloride are mixed. TEMs showstructures that are vesicular and some are multilamellar. Membranousstructures found in these TEMs are consistent with the formation oflamellar vesicular complexes in liposomes. These results show thatbenzalkonium chloride:DNA complexes form lamellar and vesicularstructures, similar to those described for liposomes and liposomalformulations.

EXAMPLE 3 Labeled DNA is Associated with the Vesicular complexes, SEMand TEM Analysis

A. Preparation of Open Circular Plasmid DNA.

Supercoiled (SC) plasmid DNA was converted into an open-circular form byheating at 80° C., for 4 hours. Nearly 80% of SC DNA was converted tonicked open circular (OC) fonn by this method. The amount of DNAconverted to OC was quantified using a video gel-scanner, followingelectrophoretic separation of SC and OC forms. The OC was purified tonearly 95% purity on a Q-Sepharose matrix using a NaCl step gradient.

B. Preparation of Bioltinylated dUTP DNA.

Fifty micrograms of nicked OC DNA was subjected to a strand displacementreaction using Klenow (the non-proofreading proteolytic fragment of DNApolymerase of E. coli), and dNTP. The dNTP mixture contained dATP, dGTP,dCTP, dUTP, and Bio-dUTP. Bio-dUTP has biotin on an 11 carbon linkerattached to the base uracil. The nucleotide concentration was at 50 μM,while the bio-dUTP was at 2 μm, and dUTP was at 20 μM. The plasmid waspurified from free nucleotides by two rounds of ethanol precipitation,and two 70% ethanol washes. The amount of biotin incorporated into theplasmid was determined by a kinetic ELISA, by reacting dilutions of theplasmid with streptavidin-horseradish peroxidase (HRP). Three moleculesof biotin were incorporated per plasmid molecule.

C. Preparation of Streptavidin-Colloidal Gold Conjugated Plasmid DNA.

One microgram of a 5 Kbp plasmid DNA corresponds to 1.25×10¹⁴ molecules(3.75×10¹³ biotin). Five hundred micrograms of mixture in one milliliterwas reacted with 10¹¹ molecules of gold-conjugated streptavidin. Thisensures absence of free streptavidin-gold conjugates [T. Daemen et al,Hepatology, 26:416 (1997)]. Benzalkonium chloride at 0.02% was added tothe mixture to form complexes. The mixture was analyzed by TEM.

D. TEM Analysis

TEM analysis of the above-prepared gold labeled DNA in thebenzylammonium-containing surfactant/polynucleic acid sequencecompositions of this invention demonstrated that the gold-labeled DNA isfound in structures (complexes) that are identical to those found bycarbon shadowing. These structures show that DNA is within the vesicularmembranes. Vesicular complexes in some fields appear to bemultilamellar, and the gold labeled plasmid molecules are interspersedwithin these lamellae. Electron diffraction shows densitiescorresponding to gold that were detected on these membranes, and withinmembranes. This analysis demonstrates that benzalkonium chloride:DNAcomplexes form vesicular complexes, and the DNA is in the vesicularspace, intimately associated with the membrane.

EXAMPLE 4 Enhancement of DNA Uptake Using Compositions of the Invention

A. The Formulations Tested

Four different formulations were prepared to evaluate the ability ofaqueous mixtures of benzylammonium-containing surfactants andpolynucleic acid sequences to facilitate DNA delivery. The fourformulations of the invention and the control are reported in Table 2below.

Formulation 1 (2 mL formulation) was prepared as follows: 0.5%benzalkonium chloride stock was prepared in 5 mM phosphate buffer. A DNAplasmid was constructed which contained the Herpes Simplex Virus geneencoding the gD₂ protein linked to a cytomegalovirus promoter and SV40polyadenylation site. This plasmid, referred to as plasmid 24, isdescribed in detail in International Patent Publication No. WO97/41892,published Nov. 13, 1997. The DNA plasmid solution was preparedcontaining 0.5 mg/mL plasmid DNA in 5 mM phosphate buffer with 8.71%sucrose. Both the benzalkonium chloride stock solution and DNA solutionwere filtered using 0.22 μm Millex GV syringe filter. Forty μL ofbenzalkonium chloride stock solution was added to 1.96 mL of DNAsolution (0.5 mg/mL) by slow mixing.

Formulation 2 (2 mL formulation) was prepared as follows: 0.5%benzalkonium chloride stock was prepared in 5 mM phosphate buffer. DNAsolution contain the above-described plasmid 24 (0.5 mg/mL) was preparedin 5 mM phosphate buffer with 8.71% sucrose. Both the benzalkoniumchloride stock solution and DNA solution were filtered using 0.22 μmMillex GV syringe filter. Eighty μL of benzalkonium chloride stocksolution was added to 1.92 mL of DNA solution (0.5 mg/mL) by slowmixing.

Formulation 3 (2 mL formulation) was prepared as follows: 0.5%benzalkonium chloride stock was prepared in 5 mM phosphate buffer. DNAsolution containing the above-described plasmid 24 (0.5 mg/mL) wasprepared in buffer solution containing 5 mM phosphate and 150 mM ofsodium chloride. Both the benzalkonium chloride stock solution and DNAsolution were filtered using 0.22 μm Millex GV syringe filter. Onehundred twenty μL of benzalkonium chloride stock solution was added to1.88 mL of DNA solution (0.5 mg/mL) by slow mixing.

As a positive control, a DNA only formulation was prepared containing0.5 mg/mL DNA of plasmid 24 in 30 mM citrate buffer, 0.1%ethylenediamine tetraacetic acid (EDTA), 150 mM NaCl at pH 6.7±0.5. As anegative control, a DNA only formulation was prepared containing 0.5mg/ml DNA of plasmid 23 (no HSV insert) in 30 mM citrate buffer, 0.1%EDTA, 150 mM NaCl at pH 6.7±0.5.

TABLE 2 Formulations Components 1 2 3 Plasmid DNA (mg/mL) 0.5 0.5 0.5Benzalkonium chloride (w/v) 0.01% 0.02% 0.06% Sucrose (w/v) 8.71% 8.71%0 Sodium Chloride (mM) 0 0 150 Phosphate buffer (mM) 5 5 5 pH 6.7 ± 0.56.7 ± 0.5 6.7 ± 0.5

B. Protocol of Test

Six groups of BALB/c mice (5 mice/group) were immunized intramuscularlywith 50 μg plasmid DNA in a total volume of 100 μL per dose, which wasdistributed between 3-4 sites per leg on Day 1. The plasmid wasadministered in compositions with different concentrations ofbenzalkonium chloride as follows:

Group I received Formulation 1 (50 μg plasmid in 0.01% benzalkoniumchloride). Group II received Formulation 2 (50 μg plasmid in 0.02%benzalkonium chloride). Group III received Formulation 3 (50 μg plasmidin 0.06% benzalkonium chloride). Group IV received positive control (50μg plasmid 24 without any transfection facilitating agent); and Group Vreceived negative control (plasmid 23 with no HSV insert and without anytransfection facilitating agent).

Serum was collected prior to first injection (Day 0) and at two weekspost injection (Day 14). Animals were boosted once more at the samedosage at four weeks (Day 28). After the final injection an additionalserum sample was taken. Two weeks following the final immunization (Day42), mice were euthanized using halothane, followed by cervicaldislocation and the spleens were harvested.

C. In Vivo Results

The sera and spleens from each mouse and each group of mice was assayedfor humorat response, i.e., antibody response to gD, as measured by astandard enzyme linked immunosorbent assay (ELISA) [J. E. Coligan et al,eds., “Current Protocols in Immunology”, Vol. 1, chap. 2.1, John Wiley &Sons, Inc. (1992)]. FIGS. 1 and 2 illustrate the humoral responses ofindividual animals and the group average humoral responses,respectively. Antibody response calculations were conducted as follows.Based on linear regression, a linear model [O.D.=(slope×antibodyconcentration)+Intercept] was fitted to the standard data. The equationfor the best fit.line was used to calculate antibody response forspecific formulations. FIG. 3 illustrates the individual and groupcellular responses of the same animals. Systemic cellular response (SI)was measured using a splenic cell proliferation assay in which wascalculated the ratio of the number of spleen cells stimulated in thepresence of HSV gD antigen and radiolabelled nucleotides divided by thenumber of the same spleen cells incubated in the absence of any antigen,but in the presence of radiolabelled nucleotides [J. E. Coligan et al,eds., “Current Protocols in Immunology”, Vol. 1, chap. 2.1, John Wiley &Sons, Inc. (1992)].

FIG. 1 demonstrates that Formulation 1 (0.01% benzalkonium chloride and0.5 mg/mL DNA) produced a significantly higher individual and grouphumoral response than did the positive control DNA. The group averagehumoral response (antibody) for Formulation 1 is 2.9 ng/mL higher thanthe positive control DNA alone formulation. FIG. 1 demonstrates that allfive animals in Groups I and II gave humoral responses to Formulations 1and 2, respectively; whereas only four animals in Group IV, whichreceived the positive control DNA, produced humoral responses. Thenegative control DNA gave no response, as predictable. Thus, greaterconsistency and predictability of immune responses was seen withformulations of this invention than with DNA delivered in the absence ofa transfection facilitating agent of this invention. Formulation 3 inthe preparation used in this experiment produced an undesirableprecipitate.

FIG. 3 demonstrates that the cellular responses for Formulations 1 and 2(Groups I and II, respectively) are comparable to those elicited byadministration of positive control DNA only (Group IV). However, again,variation in the cellular responses to formulations of this invention isminimal, compared to the wide variation in responses observed for thoseanimals in Group IV that received DNA alone.

Based on the humoral and cellular responses in animals, as describedabove, it appears that benzalkonium chloride/DNA complex formulations(Formulations 1 and 2) provide better immune responses compared to DNAalone and facilitates the transfer of DNA into the subject.

EXAMPLE 5 Benzethonium Chloride:DNA Complexes

Compositions according to the present invention were also prepared usinganother exemplary benzylammonium-containing surfactant as describedhere, i.e., benzethonium chloride. These formulations were prepared insubstantially the same manner as described for the benzalkonium chlorideformulations of Example 4 above. The different formulations appear inTable 3 below.

TABLE 3 Components Formulation 1 Formulation 2 Formulation 3 Plasmid DNA0.1 0.5 0.5 (mg/mL) Benzethonium 0.01% 0.01% 0.02% chloride (w/v)Sucrose (w/v) 8.71% 8.71% 8.71% Phosphate buffer 5 5 5 (mM) pH 6.7 ± 0.56.7 ± 0.5 6.7 ± 0.5

TEM and SEM analyses of these compositions were performed as describedin Example 3. Based on TEM and SEM analysis, the structures have thesame description as found for the benzalkonium chloride:DNA vesicularcomplexes of Example 3, i.e. uniformly sized vesicular complexes whichpackaged DNA.

All above-noted published references, and the provisional U.S. patentapplication No. 60/063,360, are incorporated herein by reference.Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the compositions and processes of the present invention are believedto be encompassed in the scope of the claims appended hereto.

What is claimed is:
 1. A soluble ionic complex consisting essentially ofan aqueous mixture of (a) a benzylammonium group-containing surfactantof the formula:

 wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³is Hor methyl; and (b) a polynucleic acid sequence.
 2. The complex accordingto claim 1, wherein X is selected from the group consisting of halide,sulfate, and carbonate.
 3. The complex according to claim 2, whereinsaid benzylammonium group-containing surfactant is a benzalkoniumhalide.
 4. The complex according to claim 3 wherein said halide ischloride and said surfactant is benzalkonium chloride.
 5. The complexaccording to claim 1, wherein said surfactant is a benzethonium halide.6. The complex according to claim 5, wherein said surfactant isbenzethonium chloride.
 7. The complex according to claim 1, wherein saidbenzylammonium containing surfactant is present in a concentration ofabout 0.001 to about 2.4% w/v.
 8. The complex according to claim 7,wherein said benzylammonium containing surfactant is present in aconcentration of about 0.001 to about 0.1% w/v.
 9. The complex accordingto claim 8, wherein said benzylammonium containing surfactant is presentin a concentration of about 0.005 to about 0.06% w/v.
 10. The complexaccording to claim 9, wherein said benzylammonium containing surfactantis present in a concentration of about 0.005 to about 0.03% w/v.
 11. Thecomplex according to claim 1, wherein said polynucleic acid sequence isa ribonucleic acid sequence.
 12. The complex according to claim 1,wherein said polynucleic acid sequence is a deoxyribonucleic acidsequence.
 13. The complex according to claim 12, wherein saiddeoxyribonucleic acid sequence is a plasmid.
 14. The complex accordingto claim 1, wherein said polynucleic acid sequence is a deoxyribonucleicacid sequence.
 15. The complex according to claim 12, wherein saidsequence encodes a protein or a peptide.
 16. The complex according toclaim 1, wherein said polynucleic acid sequence is present in saidcomplex in a concentration of between about 10 μg/ml to about 20 mg/ml.17. The complex according to claim 16, wherein said polynucleic acidsequence is present in said complex in a concentration of between about50 μg/ml to about 10 mg/ml.
 18. The complex according to claim 17,wherein said polynucleic acid sequence is present in said complex in aconcentration of between about 100 μg/ml to about 1.0 mg/ml.
 19. Acomposition consisting essentially of the complex according to claim 1and additives selected from the group consisting of buffering agents andtonicity adjusting agents.
 20. The composition according to claim 19,which is an injectable formulation.
 21. A method of inducing an immuneresponse in a mammalian subject to a viral, prokaryotic or eukaryoticpathogenic antigen comprising the step of administering by injection tocells of said subject, an effective amount of the complextion of claim19, wherein said polynucleic acid sequence comprises a sequence whichencodes at least one epitope that is identical or substantially similarto an epitope of the pathogenic antigen, said epitope-encoding sequenceunder the control of regulatory sequences that direct expression of saidprotein in the cells of said subject.
 22. The method according to claim21, wherein composition is administered by an injection route selectedfrom the group consisting of intramuscularly, intraperitoneally,intradermally, subcutaneously, intravenously, intraarterially, andintraoccularly.
 23. The complex according to claim 1, wherein saidsurfactant is benzalkonium chloride, said polynucleic acid is plasmidDNA, and said aqueous mixture is an isotonic solution.
 24. A method ofimmunizing a mammalian subject against a pathogen comprising the step ofadministering by injection to said subject an effective amount of thecomposition of claim 19, wherein said polynucleic acid sequencecomprises a nucleotide sequence encoding at least one protein epitopethat is identical to an antigen of the pathogen, said protein epitopeencoding sequence is operatively linked to regulating sequencesdirecting the expression of said protein epitope in the cells of saidsubject.
 25. The method according to claim 24, wherein said polynucleicacid sequence is administered by an injection route selected from thegroup consisting of intramuscularly, intraperitoneally, intradermally,subcutaneously, intravenously, intraarterially, and intraoccularly. 26.A composition consisting essentially of a polynucleic acid sequencesubstantially packaged in a vesicular complex formed by an aqueousmixture of a benzylammonium-containing surfactant of the formula:

wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³ is Hor methyl; and said polynucleic acid sequence.
 27. A compositionconsisting essentially of multiple vesicular complexes of uniform size,each of said vesicular complexes consisting essentially of a polynucleicacid sequence substantially packaged in a vesicular complex formed by anaqueous mixture of a benzylammonium-containing surfactant of theformula:

wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³ is Hor methyl; and said polynucleic acid sequence.
 28. The compositionaccording to claim 27 formed by mixing an aqueous solution of thesurfactant with a polynucleic acid sequence.
 29. A method of introducinga polynucleic acid sequence into a cell comprising the step ofcontacting said cell with a member of the group consisting of: (a) thecomplex of claim 1; (b) the composition of claim 26; and (C) thecomposition of claim
 27. 30. A pharmaceutical composition selected fromthe group consisting of: (a) a composition consisting essentially of apolynucleic acid sequence substantially packaged in a vesicular complexformed by an aqueous mixture of a benzylammonium-containing surfactantof the formula:

 wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³ is Hor methyl; and said polynucleic acid sequence; and (b) a compositionconsisting essentially of multiple vesicular complexes of uniform size,each of said vesicular complexes consisting essentially of a polynucleicacid sequence substantially packaged in a vesicular complex formed by anaqueous mixture of a benzylammonium-containing surfactant of theformula:

 wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³ is Hor methyl; and said polynucleic acid sequence.
 31. A method offacilitating the uptake of a polynucleic acid sequence into a cellcomprising contacting said cell with a soluble ionic complex consistingessentially of an aqueous mixture of (a) a benzylammoniumgroup-containing surfactant of the formula:

 wherein X is an anion; each R¹ is independently selected from the groupconsisting of a hydrogen and a lower alkyl group comprising from 1 to 6carbon atoms; R² is selected from the group consisting of CH₂ and —O—;R³ is selected from the group consisting of H, CH₃, C₂H₅, phenyl,mono-substituted phenyl, and di-substituted phenyl, wherein saidsubstitutions are independently selected from the group consisting ofC₁-C₁₀ branched or straight chain alkyl groups; n is an integer of 2through 7, provided that when n is 1, R³ is methyl, ethyl, phenyl orsubstituted phenyl; when n is 4 through 6, R³ is H, methyl, ethyl orphenyl; when n is 6, R³ is H, methyl or ethyl; and when n is 7, R³is Hor methyl; and (b) a polynucleic acid sequence.
 32. The method accordingto claim 31, wherein said complex is in the form of a vesicular complexin which the polynucleic acid sequence is substantially packaged. 33.The complex according t o claim 1, which forms a vesicular complex inwhich the polynucleic acid sequence is substantially packaged.